PROJECT SUMMARY/ABSTRACT This NIH K08 proposal describes a five-year training program for career development in academic neuropathology and developmental neurogenetics. Dr. Matthew Rose has completed clinical residency in Anatomic Pathology and fellowship in Neuropathology at Brigham and Women?s Hospital (BWH) and Boston Children?s Hospital (BCH) at Harvard Medical School (HMS), and will embark on a research program designed to train for an independent academic career in neurologic disease-oriented research. In this training program, Dr. Rose will acquire in-depth experience in the study of ocular motor neuron (OMN) development and axon targeting, interpretation of single cell RNA-sequencing, analysis of gene regulatory networks, tissue clearing and advanced imaging approaches, and gene-editing technologies. His mentor, Dr. Elizabeth Engle (a Professor of Neurology and HHMI Investigator at BCH), is a leader in brainstem OMN development and congenital cranial dysinnervation disorders (CCDDs) that involve these motor neurons. Her laboratory has extensive experience in human and mouse genetics, neuroanatomy, large data analyses, molecular biology, imaging, and cell culture techniques. She has an established track record for mentoring other trainees to successful careers in biomedical investigation. In addition to his mentor, he has assembled a group of collaborators with complementary expertise in these disciplines, and an Advisory Committee of senior faculty with extensive experience in guiding physician-scientists through the transition to independence. He will supplement this training with didactics and presentation of work at national meetings. The primary scientific goal of the proposed research plan is to study normal and abnormal OMN specification and axonal growth and guidance in health and disease. Dr. Rose provides an initial map of the genetic differences among brainstem motor neurons that may serve as a foundation for further studies of all motor neuron diseases. These preliminary data will be used as a foundation to further study the development of the OMN subnuclei by discovering markers of each OMN subpopulation, and then applying those markers to the study of the CCDDs. The central hypothesis of this proposal is that the differences in gene expression among OMN subpopulations may predispose certain subtypes to undergo dysinnervation in different CCDDs. Single cell RNA-seq will be performed on the three primary OMNs: the oculomotor, trochlear, and abducens brainstem nuclei, in order to discover markers of each subpopulation. In combination with ATAC-seq, the transcriptional regulatory networks for OMN specification will be investigated. Unique markers of each OMN subpopulation will then be used to study normal OMN axon projections to distinct muscle targets, as well as using them to dissect the contributions of different populations to the stereotypic and pathologic aberrant innervations observed in the CCDDs. These studies have significance to the broader fields of neuronal specification and differentiation and to axonal growth and guidance.